Generation of DG75 B cells lacking expression of PIP5Kα and PIP5Kγ In Vav1-deficient DG75 B cells, both isoforms, PIP5Kα and PIP5Kγ showed the same potency

in enhancing BCR-induced Ca²⁺-mobilization (Figure 4.19). I used the previously generated and characterized PIP5Kα-deficient DG75 clones #13 and #25 for nucleofection with plasmids targeting either exon 3 or exon 4 of the PIP5K1C gene for CRISPR/Cas9-mediated genome editing. The resulting single cell sub-clones originating from PIP5Kα-deficient clone #25 were screened for expression of PIP5Kγ using specific antibodies (Figure 4.23 A). Furthermore, the obtained clones lacking the expression of both, PIP5Kα and PIP5Kγ (Figure 4.23 B, C and D) were genetically characterized and checked for their ability to mobilize Ca²⁺ upon stimulation of the BCR (Figure 4.23 E). Genetic analysis of PIP5Kα-deficient clone #25 derived clones #1 and #5 lacking the expression of PIP5Kγ (Figure 4.23 A) revealed that in each clone both mutant alleles led to early frame shifts resulting in premature stop codons (Figure 4.23 C and D). However, the capacity to mobilize Ca²⁺ upon stimulation of the BCR was not altered for double-deficient DG75 B cells (Figure 4.23 E, turquoise and grey lines) compared to their parental precursor #25 with no expression of PIP5Kα (Figure 4.23 E, red line).

Figure 4.23 Targeting exon 3 of PIP5KC successfully generated double-deficient DG75 B cells for PIP5Kα and PIP5Kγ. (A) Immunoblot analysis of cleared cellular lysates of DG75 B cell sub-clones transfected with plasmids to CRISPR PIP5Kγ. Proteins were separated by SDS-PAGE and analyzed by western blot using antibodies specific for PIP5Kγ and Actin. The molecular weight of marker proteins is indicated on the left in kDa.

(B) Restriction analysis of the amplicon covering exon 3 of PIP5K1C. The amplicon was amplified by PCR, the resulting DNA was separated by agarose gel electrophoresis and extracted from the gel. Half of the DNA was left untreated whereas the other half was incubated with TaqI. After this incubation period, the DNA was separated on an agarose gel and was visualized using ethidium bromide. The size of marker DNA is indicated on the left. (C and D) Sequencing data of exon 3 of PIP5K1C for clones #1 and #5. The amplicon was amplified by PCR, the resulting DNA was separated in an agarose gel and extracted from the gel. The DNA was cloned into the pCR2.1 plasmid and the insert was sequenced. Differences to the wild-type sequences are highlighted in red. (E) Analysis of intracellular BCR-induced Ca²⁺-mobilization for DG75 B cells lacking the expression of PIP5Kα and PIP5Kγ. Cells were loaded with the Ca²⁺-sensitive fluorophore Indo-1 AM. After recording of basal Ca²⁺-levels for 25 sec, cells were stimulated with 20 µg/ml of anti-human-IgM F(ab’)2 fragments (α IgM) and the fluorescence was monitored for a total time of 5 min.

I repeated the same procedure to generate double-deficient cells for PIP5Kα and PIP5Kγ originating from PIP5Kα-deficient cell clone #13, this time targeting exon 3 of PIP5K1C (Figure 4.24).

Figure 4.24 Targeting exon 3 of PIP5KC successfully generated double-deficient DG75 B cells for PIP5Kα and PIP5Kγ. (A) Immunoblot analysis of cleared cellular lysates of DG75 B cell sub-clones transfected with plasmids to CRISPR PIP5Kγ. Proteins were separated by SDS-PAGE and analyzed by western blot using antibodies specific for PIP5Kγ and Actin. The molecular weight of marker proteins is indicated on the left in kDa.

(B) Restriction analysis of the amplicon covering exon 3 of PIP5K1C. The amplicon was amplified by PCR, the resulting DNA was separated by agarose gel electrophoresis and extracted from the gel. Half of the DNA was left untreated whereas the other half was incubated with TaqI. After this incubation period, the DNA was separated on an agarose gel and was visualized using ethidium bromide. The size of marker DNA is indicated on the left. (C) Sequencing data of exon 3 of PIP5K1C for clone #3. The amplicon was amplified by PCR, the resulting DNA was separated in an agarose gel and extracted from the gel. The DNA was cloned into the pCR2.1 plasmid and the insert was sequenced. Differences to the wild-type sequences are highlighted in red. (D) Analysis of intracellular BCR-induced Ca²⁺-mobilization for DG75 B cells lacking the expression of PIP5Kα and PIP5Kγ. Cells were loaded with the Ca²⁺-sensitive fluorophore Indo-1 AM. After recording of basal Ca²⁺-levels for 25 sec, cells were stimulated with 20 µg/ml of anti-human-IgM F(ab’)2 fragments (α IgM) and the fluorescence was monitored for a total time of 5 min.

Genetic characterization (Figure 4.24 B and C) of the obtained PIP5Kα and PIP5Kγ double-deficient knockout clone #3 (Figure 4.24 A) revealed early frame shifts resulting in premature stop codons on both alleles. However, as before, the clone showed the same ability to mobilize Ca²⁺ upon stimulation of the BCR (Figure 4.24 D, olive line) compared to its PIP5Kα-deficient precursor #13 (Figure 4.24 D, orange line).

To verify the successful genetic inactivation of PIP5Kα and PIP5Kγ in the generated knockout clones and to investigate a possibly undesired upregulation of PIP5Kβ expression, western blot analyses were conducted for each of the PIP5K family members (Figure 4.25).

Figure 4.25 PIP5Kα and PIP5Kγ double-deficient DG75 B cells do not express any member of the PIP5K family. Immunoblot analysis of cleared cellular lysates of parental DG75 B cells or DG75 B cells deficient for either Vav1, PIP5Kα (α-ko) or PIP5Kα and PIP5Kγ (γ-ko). Proteins were separated by SDS-PAGE and analyzed by western blot using antibodies specific for PIP5Kα, PIP5Kβ, PIP5Kγ and Actin. The molecular weight of marker proteins is indicated on the left in kDa. As a positive control for antibodies to PIP5Kβ (+ (β)), lysates from Vav1-deficient DG75 B cells with induced expression of Citrine-tagged PIP5Kβ were used.

None of the three generated PIP5Kα and PIP5Kγ double-deficient DG75 B cell clones (Figure 4.25, lanes 5 to 7), either originating from PIP5Kα-deficient clone #13 (Figure 4.25, lane 3) or from clone #25 (Figure 4.25, lanes 4), expressed detectable amounts of PIP5K family members, thus most likely representing total PIP5K-negative cells.

4.4.4. Generation of DG75 cells deficient for all three members of the PIP5K